I have read many papers that use chemiluminescent detection, mostly using HRP conjugated antibodies for dot-blot. But I don't have the necessary equipment to measure the signal, and I've heard the process is very time-consuming. I've read some protocols for fluorescent dot-blot assays and have found some suitable antibodies but have not found any papers referencing these methods. I was hoping to use this method since it seems a lot quicker and requires less materials. I am hoping to measure the signal using a fluorescence microplate reader, but am wondering if the signal would be reproducible.
Hi Erin, quantitative measurements using fluorescence are problematic because environmental factors influence how well a fluorophore works and you don't know how many flurophores are on antibodies.
You could do serial dilutions of the control fluorescent Ab on the plates as a standard curve to evaluate your samples. This is semi-quantitative at best.
I think you would be better off with an ELISA instead of a dot blot. ELISAs are capable of quantitative measurements by absorbance, fluorescence, or chemiluminescence using a suitable plate reader. For fluorescence you should use a black plate.
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